Transcription activator-like effectors (TALEs) are important genome science tools (see, e.g., Campbell et al., Circ. Res., 113:571-587 (2013); U.S. Pat. No. 8,748,134 issued on Jun. 10, 2014; U.S. Pat. Publication No. US 2011/0145940; Cermak et al., Nucleic Acids Res. 2011 July; 39(12):e82. doi: 10.1093/nar/gkr218), including an array of applications from locus-specific DNA editing, artificial transcriptional activators and repressors, locus-specific epigenetic modification, or live subcellular imaging. TALEs as nucleases (TALENs) are popular genome editing tools that are extensively deployed in both in vitro cell systems and diverse model organisms. Native to the plant pathogen Xanthomonas, TALEs normally function as transcription factors. TALEs were harnessed in molecular biology applications to target almost any DNA sequence and effect changes to nearby DNA and genes when fused to other proteins such as endonucleases or activator and repressor domains. The DNA recognition sequence of the TALE is typically a repetitive tract encoding 33-35 amino acid residues, with the last repetitive module being a partial or half (20 amino acids) length. Two variable, adjacent positions, 12 and 13, within this repetitive sequence (known as repeat-variable di-residues, or RVDs) are responsible for distinguishing between the four DNA nucleotides (Moscou and Bogdanove, Science, 326:1501-1508 (2009); and Boch et al., Science, 326:1509-1512 (2009)). Commonly used RVDs, each preferentially targeting a specific nucleotide, include: NI, NN=adenine; HD=cytosine; NK, NN, NH=guanine, NG=thymine.
By fusing multiple RVD modules in a specific order, a TALE can be targeted to a corresponding linear DNA sequence. Diverse protocols have been developed to efficiently assemble these repeat modules. The Golden Gate TALE system (Golden Gate TALEN and TAL Effector Kit 2.0, referred to herein as GGT Kit) is a popular assembly method (Cermak, et al., Nucleic Acids Res., 39:e82 (2011)). This is a 3-step, 5-day Golden Gate assembly process deployed in many laboratories thanks in part to its flexibility, low start-up cost, and requirement of small number of common molecular cloning reagents. Several high throughput TALEN assembly methods (Reyon et al., Nat. Biotechnol., 30:460-465 (2012); Briggs et al., Nucleic Acids Res., 40:e117 (2012); Wang et al., Angew. Chem. Int. Ed. Engl., 51:8505-8508 (2012); Sanjana et al., Nat. Protoc, 7:171-192 (2012); Schmid-Burgk et al., Nat. Biotechnol., 31:76-81 (2012); Sakuma et al., Genes Cells, 18:315-326 (2013); Uhde-Stone et al., Biol. Proced. Online, 15:3 (2013); and Ding et al., Cell Stem Cell, 12:238-251 (2013)) offer unique advantages over the GGT kit and include features like automation, but each of these systems also come with specific limitations. For example, the lowest supply cost assembly system reported to date requires the purchase of a liquid handling robot (Liang et al., ACS Synth. Biol, 3:67-73 (2014)).